Prediction of functional consequences of the five newly discovered G6PD variations in Taiwan.

Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency; OMIM #300908) is the most common inborn error disorders worldwide. While the G6PD is the key enzyme of removing oxidative stress in erythrocytes, the early diagnosis is utmost vital to prevent chronic and drug-, food- or infection-induced hemolytic anemia. The characterization of the mutations is also important for the subsequent genetic counseling, especially for female carrier with ambiguous enzyme activities and males with mild mutations. While multiplex SNaPshot assay and Sanger sequencing were performed on 500 G6PD deficient males, five newly discovered variations, namely c.187G > A (p.E63K), c.585G > C (p.Q195H), c.586A > T (p.I196F), c.743G > A (p.G248D), and c.1330G > A (p.V444I) were detected in the other six patients. These variants were previously named as the Pingtung, Tainan, Changhua, Chiayi, and Tainan-2 variants, respectively. The in silico analysis, as well as the prediction of the structure of the resultant mutant G6PD protein indicated that these five newly discovered variants might be disease causing mutations.

Applying a multiplexed primer extension method on dried blood spots increased the detection of carriers at risk of glucose-6-phosphate dehydrogenase deficiency in newborn screening program.

BACKGROUND: Patients with glucose-6-phosphate dehydrogenase deficiency might develop acute hemolytic anemia, chronic hemolytic anemia, and neonatal hyperbilirubinemia when exposed to high levels of oxidative stress. Severe hemolysis may occur in not only patients but also female carriers under certain conditions. However, 80%-85% of female carriers were undetected in an existing newborn screening program because of their wide-ranging levels of enzyme activity. METHODS: We developed a cost- and time-efficient multiplex SNaPshot assay using dried blood spots. RESULTS: By detecting 21 common mutations in Taiwan and Southeast Asia, the assay could determine 98.2% of the mutant alleles in our cohort of Taiwanese newborns. The 9 undetermined mutant alleles were consequently detected by Sanger sequencing, of which 5 unpublished variations-c.187G > A (Pingtung), c.585G > C (Tainan), c.586A > T (Changhua), c.743G > A (Chiayi), and c.1330G > A (Tainan-2)-were detected. Furthermore, 13% of mild mutations were missed in male infants whose enzyme levels at 6.1-7.0 U/gHb in the newborn screening program when set the cutoff value at 6.0 U/gHb. We therefore suggest increasing the cutoff value and applying the multiplex SNaPshot assay as the second tier for neonatal screening. CONCLUSIONS: Our approach could significantly increase the detection rate of male patients and female carriers with a reasonable cost and a reasonable number of clinic referrals.

Biphasic and Stage-Associated Expression of CPEB4 in Hepatocellular Carcinoma.

Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is a sequence-specific RNA-binding protein and translational regulator, with expression associated with tumor progression. Nevertheless, CPEB4 seems to play paradoxical roles in cancers-an oncogenic promoter in pancreatic ductal adenocarcinoma (PDA) and glioblastomas but a tumor suppressor in hepatocellular carcinoma (HCC). To assess whether CPEB4-regulated carcinogenesis is tissue-specific, we reevaluated the role of CPEB4 in HCC. Although proliferation of hepatocytes appeared normal in CPEB4-knockout (KO) mice after partial hepatectomy, knockdown (KD) of CPEB4 in HepG2 liver cancer cells promoted colony formation in vitro. Moreover, the growth of CPEB4-KD cells was accelerated in an in vivo xenograft mouse model. In tumorous and adjacent non-tumorous paired liver specimens from 49 HCC patients, the protein level of CPEB4 was significantly upregulated in early-stage HCC but decreased toward late-stage HCC. This finding agrees with changes in CPEB4 mRNA level from analysis of two sets of HCC microarray data from the Gene Expression Omnibus (GEO) database. Taken together, downregulation of CPEB4 likely occurs at the late cancer stage to facilitate HCC progression. Biphasic alteration of CPEB4 expression during HCC progression suggests its complicated role in tumorigenesis.

Endothelial Cell Sensitization by Death Receptor Fractions of an Anti-Dengue Nonstructural Protein 1 Antibody Induced Plasma Leakage, Coagulopathy, and Mortality in Mice.

The mechanisms leading to the life-threatening dengue hemorrhagic fever (DHF) remain elusive. DHF preferentially occurs during secondary dengue infections, suggesting that aberrant immune responses are involved in its development. We previously demonstrated that the autoantibodies elicited by dengue virus (DENV) nonstructural protein 1 (NS1; anti-NS1 Igs) induce plasma leakage and mortality in mice with warfarinized anticoagulant suppression. However, the involved pathogenic Ig fractions of anti-NS1 Igs remain unclear. In this study, the autoreactive Igs in patients with DHF and in NS1-immunized rabbits crossreacted with TNF-related apoptosis-inducing ligand receptor 1 (death receptor [DR]4). Challenges with the DENV in a subcytotoxic dose sensitized endothelial cells to apoptosis. Treatments with the autoantibodies induced proapoptotic activities and suppressed the surface expression of endothelial anticoagulant thrombomodulin. Combined treatments comprising the DENV and DR4 affinity-purified fractions of anti-NS1 IgGs (anti-NS1-DR4 Ig), but not preimmune control IgGs, in subcytotoxic doses led to apoptosis in endothelial cells. Treatments with the anti-NS1-DR4 Ig led to plasma leakage, coagulopathy, and morality in mice with warfarinized anticoagulant suppression. These results suggest that DR4-induced endothelial cell sensitization through NS1-elicited autoantibodies exacerbates anticoagulant suppression, vascular injury, and plasma leakage. Detecting and blocking anti-DR Igs in patients may be novel strategies for managing severe DENV infection.

Two frequent mutations associated with the classic form of propionic acidemia in Taiwan.

Propionyl-CoA carboxylase (PCC) is involved in the catabolism of branched chain amino acids, odd-numbered fatty acids, cholesterol, and other metabolites. PCC consists of two subunits, α and ß, encoded by the PCCA and PCCB genes, respectively. Mutations in the PCCA or PCCB subunit gene may lead to propionic acidemia. In this study, we performed mutation analysis on ten propionic acidemia patients from eight unrelated and nonconsanguineous families in Taiwan. Two PCCA mutations, c.229C→T (p.R77W) and c.1262A→C (p.Q421P), were identified in a PCCA-deficient patient. Six mutations in the PCCB gene, including c.-4156_183+3713del, c.580T→C (p.S194P), c.838dup (p.L280Pfs 11), c.1301C→T (p.A434V), c.1316A→G (P.Y439C), and c.1534C→T (p.R512C), were identified in seven PCCB-deficient families. The c.-4156_183+3713del mutation is the first known large deletion that affects the PCCB gene functions. Furthermore, the c.1301C→T and c.-4156_183+3713del mutations in the PCCB gene have not been reported previously. Clinical features demonstrated that these two frequent mutations are associated with low enzyme activity and a classic propionic acidemia phenotype.

Mutation spectrum of and founder effects affecting the PTS gene in East Asian populations.

The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTPS, gene symbol: PTS) is involved in the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), which is a vital cofactor of nitric oxide synthases and three types of aromatic amino acid hydroxylases; the latter are important enzymes in the production of neurotransmitters. We conducted a study of PTS mutations in East Asia, including Taiwan, Mainland China, Japan, South Korea, the Philippines, Thailand and Malaysia. A total of 43 mutations were identified, comprising 22 previously reported mutations and 21 new discovered mutations. Among these, the c.155A>G, c.259C>T, c. 272A>G, c.286G>A and c.84-291A>G mutations were the most common PTS mutations in East Asia, while the c.58T>C and c.243G>A mutations were, respectively, specific to Filipinos and Japanese originating from Okinawa. Further studies demonstrated that each of the mutations listed above was in linkage disequilibrium to a specific allele of polymorphic microsatellite marker, D11S1347. These results suggest the presence of founder effects that have affected these frequent mutations in East Asia populations. In this context, D11S1347 should become one of the most reliable polymorphic markers for use in prenatal diagnosis among PTPS deficient families, especially where mutations are yet to be identified.

Mutation Profile of the MUT Gene in Chinese Methylmalonic Aciduria Patients.

The mut-type methylmalonic aciduria (MMA, MIM 251000) is caused by a deficiency of mitochondrial methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2) activity, which results from defects in the MUT gene. To elucidate the mutation spectrum of the MUT gene in Chinese MMA patients, 13 exons of the MUT gene, including untranslated regions, were analyzed by PCR-based sequencing for 42 unrelated Chinese MMA patients. All the 42 patients were found to have at least one MUT mutation. A total of 41 mutations were identified. Of these mutations, 20 were novel ones, including one nonsense mutation (c.103C>T), 12 missense mutations (c.316A>C, c.424A>G, c.494A>G, c.554C>T, c.599T>C, c.919T>C, c.1009T>C, c.1061C>T, c.1141G>A, c.1208G>A, c.1267G>A, and c.1295A>C), one duplication (c.755dupA), three small deletions (c.398_399delGA, c.1046_1058del, and c.1835delG), two mutations that might affect mRNA splicing (c.754-1G>A and c.1084-10A>G), and one major deletion. Among the mutations identified, the c.1280G>A (15.5%), c.729_730insTT (10.7%), c.1106G>A (4.8%), c.1630_1631GG>TA (4.8%), and c.2080C>T (4.8%) accounted for 40% of the diseased alleles. The c.1280G>A and c.729_730insTT mutations were found to be the most frequent mutations in Southern and Northern Chinese, respectively. The results of microsatellite analysis suggest that the spread of c.729_730insTT among the Northern Chinese and of c.1280G>A and c.1630_1631GG>TA among the Southern Chinese may have undergone founder effects. This mutation analysis of the gene responsible for mut-type MMA will help to provide a molecular diagnostic aid for differential diagnosis of MMA and could be applied for carrier detection and prenatal diagnosis among Chinese family at risk of mut-type MMA.

Expression and significance of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase in non-melanoma skin cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression and significance of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-2, TIMP-1) in non-melanoma skin cancer (NMSC).</p><p><b>METHODS</b>Thirty six patients with squamous cell carcinoma (SCC) and 32 patients with basal cell carcinoma (BCC), confirmed by pathology, were selected, and 30 cases of normal skin were selected as control. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in all samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The expression rate, expression intensity and expression level of each factor were recorded. The results were compared between the groups.</p><p><b>RESULTS</b>The expression rates of MMP-2 and MMP-9 in the control group were 30.0% and 36.7%, the expression levels of MMP-2 and MMP-9 in the control group were 57.216 ± 12.785 and 59.318 ± 13.262, all significantly lower than those in the tumor edge and center of the SCC and BCC groups (P < 0.01). The expression rates of TIMP-1 and TIMP-2 in the control group were 96.7% and 100%, their expression levels were 121.738 ± 25.516 and 122.612 ± 25.964, all significantly higher than those in the SCC and BCC groups (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor center and edge of SCC group were significantly higher than those in the corresponding parts of the BCC group, while the expression levels of TIMP-1 and TIMP-2 were significantly lower than those in the BCC group (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor edge of the SCC and BCC groups were significantly higher than those in the tumor centers (P < 0.01), while the expression levels of TIMP-1and TIMP-2 were significantly lower than those in the tumor centers (P < 0.01).</p><p><b>CONCLUSION</b>MMP-2, MMP-9 and TIMP-2, TIMP-1 may play an important role in the development, progression, invasion and metastasis of non-melanoma skin cancer.</p>

Mutation spectrum of MMACHC in Chinese patients with combined methylmalonic aciduria and homocystinuria.

The cblC type of combined methylmalonic aciduria (MMA) and homocystinuria (HC) is the most common inborn error of vitamin B(12) metabolism and is caused by mutations in the MMACHC gene. To elucidate the spectrum of mutations that causes combined MMA and HC in Chinese patients, the MMACHC gene was sequenced in 79 unrelated Chinese patients. Sequence analysis identified 98.1% of disease alleles and found that all patients had at least one MMACHC mutation. A total of 24 mutations were identified. Out of the 24 mutations identified, 9 were novel ones, including missense mutations (c.365A>T and c.452A>G), nonsense mutations (c.315C>G and c.615C>A), deletions (c.99delA and c.277-3_c.303del30), duplications (c.248dupT and c.626dupT) and an insertion (c.445_446insA). The c.609G>A, c.658_660delAAG, c.482G>A, c.394C>T and c.80A>G mutations were the most common mutations and accounted for 80% of disease alleles. Haplotype analysis suggests that the spread of the c.80A>G, c.609G>A and c.658_660delAAG mutations in Chinese patients were caused by a founder effect. The results indicate that defects occurring in the MMACHC gene are the major cause of this disease in Chinese patients with combined MMA and HC, and direct mutation analysis can therefore be used as a rapid confirmatory diagnosis among these Chinese patients.

Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke.

Murine olfactory ensheathing cells (OECs) promote central nervous system axonal regeneration in models of spinal cord injury. We investigated whether OECs could induce a neuroplastic effect to improve the neurological dysfunction caused by hypoxic/ischemic stress. In this study, human OECs/olfactory nerve fibroblasts (hOECs/ONFs) specifically secreted trophic factors including stromal cell-derived factor-1alpha (SDF-1alpha). Rats with intracerebral hOEC/ONF implantation showed more improvement on behavioral measures of neurological deficit following stroke than control rats. [18F]fluoro-2-deoxyglucose PET (FDG-PET) showed increased glucose metabolic activity in the hOEC/ONF-treated group compared with controls. In mice, transplanted hOECs/ONFs and endogenous homing stem cells including intrinsic neural progenitor cells and bone marrow stem cells colocalized with specific neural and vascular markers, indicating stem cell fusion. Both hOECs/ONFs and endogenous homing stem cells enhanced neuroplasticity in the rat and mouse ischemic brain. Upregulation of SDF-1alpha and CXCR4 in hOECs/ONFs promoted neurite outgrowth of cocultured primary cortical neurons under oxygen glucose deprivation conditions and in stroke animals through upregulation of cellular prion protein (PrP C) expression. Therefore, the upregulation of SDF-1alpha and the enhancement of CXCR4 and PrP C interaction induced by hOEC/ONF implantation mediated neuroplastic signals in response to hypoxia and ischemia.

Enhancement of neuroplasticity through upregulation of beta1-integrin in human umbilical cord-derived stromal cell implanted stroke model.

Neuroplasticity subsequent to functional angiogenesis is an important goal for cell-based therapy of ischemic neural tissues. At present, the cellular and molecular mechanisms involved are still not well understood. In this study, we isolated mesenchymal stem cells (MSCs) from Wharton's jelly (WJ) to obtain clonally expanded human umbilical cord-derived mesenchymal stem cells (HUCMSCs) with multilineage differentiation potential. Experimental rats receiving intracerebral HUCMSC transplantation showed significantly improved neurological function compared to vehicle-treated control rats. Cortical neuronal activity, as evaluated by proton MR spectroscopy (1H-MRS), also increased considerably in the transplantation group. Transplanted HUCMSCs migrated towards the ischemic boundary zone and differentiated into glial, neuronal, doublecortin+, CXCR4+, and vascular endothelial cells to enhance neuroplasticity in the ischemic brain. In addition, HUCMSC transplantation promoted the formation of new vessels to increase local cortical blood flow in the ischemic hemisphere. Modulation by stem cell-derived macrophage/microglial interactions, and increased beta1-integrin expression, might enhance this angiogenic architecture within the ischemic brain. Inhibition of beta1-integrin expression blocked local angiogenesis and reduced recovery from neurological deficit. In addition, significantly increased modulation of neurotrophic factor expression was also found in the HUCMSC transplantation group. In summary, regulation of beta1-integrin expression plays a critical role in the plasticity of the ischemic brain after the implantation of HUCMSCs.